Many fundamental studies on primary human tissues are hampered by the paucity of obtainable substrates. Even though cell-based therapy holds great promises for the treatment of a number of hereditary as well as acquired disorders, it will mostly remain a theoretical prospect unless the requirements for providing a high number of therapeutic cells can be met. As compared to the controlled expansion of human embryonic-induced pluripotent (progenitor) stem cells, with their concomitant risks of potential tumourigenic outcomes, the expansion represents an attractive alternative for producing large quantities of cells for either experimental or therapeutic purposes.
In the most desirable scenario, cells harvested in vivo are induced to grow indefinitely in vitro, a process referred to as immortalization. Furthermore, the primary phenotype and physiological activity of the cells, including their non-dividing status if relevant, can be restored at will. Cell lines therefore obtained can be expanded, extensively characterized both in their immortalized and “de-immortalized” (i.e., after removal/silencing of the telomere length maintenance genes) states. They can be used for analyses that range from basic physiology to proteomics (of low-abundance proteins), for the production of specific proteins, toxicity testing, drug-drug interactions, drug discovery and, in selected cases, for transplantation (personalized medicine).
As most primary cells do not grow indefinitely in culture, there is need for methods to reversibly immortalize primary cells and to overcome the eventual senescent state of these cells. After a series of population doublings, the number of which varies by species, cell type, culture conditions, cells that enter a replication senescent state will no longer divide. This state makes it that normal human cells exhibit a limited replicative lifespan. The latter is characterized by the progressive intracellular accumulation of one or another of a series of tumour suppressor proteins which inhibit G1 cyclin-dependent kinases to phosphorylate p53 and pRb, two transcription regulation proteins.
Prolonged exit from the cell cycle and arrest in G1 is marked by distinct changes in cell morphology, appearance of pycnotic granules, progressive vacuolisation, alterations in gene expression and decreased telomere length which eventually lead to cell death.
Some primary cells become spontaneously immortalized and escape replication senescence. Most of the time, they have genetic mutations which makes them less a reliable representative of their starting tissue's phenotype.
Another mechanism that is used to momentarily overcome senescence is the inhibition of telomere shortening. Telomeres are repetitive “TTAGGG” stretches of DNA at the very end of the linear chromosomes. Their specific conformation avoids the chromosome being recognized as a break and protects the ends from exonuclease degradation and telomeres fusions. Telomerase Reverse Transcriptase (TERT) will lengthen the telomeres by de novo synthesis of telomeric repeats ensuring telomere length homeostasis. Ectopic expression of the human TERT (hTERT) gene has been used to induce momentary immortalization in some primary cells. However, many lines of evidence suggest that activation of the telomerase is not sufficient for immortalization of most cell types and immortalization must in such cases be achieved by introduction of viral genes such as the SV40 large T antigen. Salmon et al. (2000) describe a lentiviral approach to induce immortalization in senescent cells by introducing both hTERT and SV40 in the cell (Salmon et al., 2000, Mol. Ther., (4):404-414). However, even though introduction of viral genes such as the Epstein Barr Virus (EBV), Simian virus 40 (SV40), large T antigen (Tag), Adenovirus E1A and E1B, human papilloma virus (HPV) E6 and E7 have been equally used to permanently immortalize primary cells, such immortalized cells lose the properties of primary cells by inactivating one or another (depending on the cell type) of the above mentioned tumour suppressor genes, which allow cells to re-enter the cell cycle and permanently bypass replicative senescence. CHO, human Hela and PER-C6 cells are examples of such established cell lines.
It has also been shown that under routine cell culture conditions, many primary cells progressively increase their intracellular levels of a tumour suppressor leading to their exit from the cell cycle and accumulation somewhere in G1. In some cell types, tumour suppressor proteins appear to be a key requirement for efficient immortalization by hTERT. This suggests that immortalization of human primary cells should be achievable through the concomitant ectopic expression of hTERT and depletion of the cognate tumour suppressor protein.
Haga et al. (2006) have described the immortalization of primary cells by ectopically introduction of an hTERT enzyme and simultaneously repressing p16INK4A by a p16INK4A shRNA and introduction of Bmi-1 (Haga et al., 2007, Cancer Science, 98:147-154). Therefore, retroviral vectors were cloned and introduced in the cell by means of retroviral transfection. The authors found that cells which simultaneously repress p16 and express hTERT were able to become permanently immortalized.
WO 2 010 000 491 discloses an inducible method and one or more transgenes to induce either immortalization or senescence in primary cells, by introducing at least one immortalizing gene sequence and one sequence to down-regulate a tumour suppressive gene. The cassettes are introduced by a lentiviral system, which introduces the transgenes into the genome of the host cell at random sites.
The conventional vectors currently used in the process of primary cell immortalization rely either on random integration in the host genome or are only transiently retained.
There remains a need in the art for an improved method of inducing immortalization in primary cells. Both create serious problems with respect to safety, reproducibility and efficiency. Random integration may lead to insertional mutagenesis and to silencing of the transgene. Transient expression, on the other hand, implies that repeated treatments would be necessary and this in most cases is not desirable. Therefore the ideal vector should be retained in many cells without integration. A number of virus-based vectors replicate episomally in some mammalian cells. However, since replication of these constructs relies on the presence of virus-encoded trans-acting factors, which often lead to cell transformation, their use for genetic manipulation of eukaryotic cells is largely limited.
Therefore, there is a need for an alternative method to induce immortalization in primary cells, which preferentially has a low immunogenicity, which is devoid of the risk of random insertional mutagenesis (retroviruses) and epigenetic silencing of the introduced vector or transgene. Furthermore, it is desirable that the vectors carrying the shRNA and hTERT cassettes be maintained in both dividing and non-dividing cells. Finally, the method should equally allow for the reversibility of the state of the cells, that is, it should be possible to reverse the state of a cell, immortalized by the present invention, to its initial state, that is its primary quiescent fully differentiated state.
The invention thereto aims to provide a method for immortalizing human primary cells in a controlled and stable manner. The invention therefore discloses a transgenic system comprising one or more transgenes which can be obtained in large amounts by prior amplification in bacteria, which comprises cassettes that upon exogenous induction will activate telomere length homeostasis and cell cycle traverse i.e. immortalization of the primary host cell. The transgenic system is designed in such a way that it remains episomal and will not integrate in the host genome. This will avoid the risk of random insertional mutagenesis and its consequences. Furthermore, the designed transgenic system is not dependent on viral components for its replication in the host cell, therefore eliminating the potential risk of cell transformation. The present invention furthermore provides for a mechanism whereby the state of the immortalized cells can be reversed to its original replication senescent state without altering the genetic and phenotypic background of the primary cell.